Degradation of proteins is highly specific and tightly regulated by energy-dependent compartmental proteases in all prokaryotic and eukaryotic cells. These enzymes, members of the AAA+ ATPase family, use ATP hydrolysis to drive the mechanical unfolding of protein substrates and their translocation into a sequestered degradation chamber. The major ATP-dependent protease in eukaryotic cells is the 26S proteasome, which controls protein homeostasis and numerous vital processes by specifically degrading regulatory proteins involved for instance in transcription, cell-cycle control, signal transduction, and apoptosis. Most proteasomal substrates are marked for degradation by the reversible attachment of a poly-ubiquitin chain, which acts as a tethering signal for substrate delivery. Substantial knowledge about ubiquitin-tagging and de-ubiquitinating systems is already available, but only very little is known about the detailed mechanisms that control substrate degradation by the proteasome. The long-term objective of this proposal is to understand the molecular bases for substrate recognition, ATP-dependent forceful unfolding and translocation, and the regulation thereof by de- ubiquitination and fine-tuning of the proteasomal unfolding machinery. My lab has devised novel systems for the heterologous expression of the proteasomal 19S base in E.coli and insect cells, and the reconstitution of functional 26S proteasomes in vitro. This provides us with powerful tools for extensive mutagenesis and unprecedented mechanistic studies. Using a combination of biochemical and biophysical approaches, our goals are to 1) further develop the eukaryotic 26S proteasome for quantitative in-vitro analyses, 2) determine the molecular mechanisms underlying coordinated ATP-hydrolysis, substrate interactions, and the timing of de-ubiquitination, and 3) understand the mechano-chemical coupling and the generation of unfolding force. We anticipate that our results will contribute to the general understanding of ATP-dependent molecular machines, ubiquitin signaling, and the regulation of protein turnover in eukaryotic cells, and thus impact several different areas of biochemistry, molecular biology, and cell-biological research. Given the role of the proteasome in the pathogenesis of numerous human diseases, a detailed knowledge of the molecular mechanisms for substrate processing has also significant biomedical relevance and may aid the development of novel, more specific drugs targeting the 26S proteasome. PUBLIC HEALTH RELEVANCE: The eukaryotic 26S proteasome is a proteolytic molecular machine that regulates many vital processes in the cell. This proposal aims to understand in molecular detail the biochemistry, mechanics, and regulation of substrate recognition, ATP-dependent unfolding, and processing by the proteasome.